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Regulation of Cytoplasmic Dynein by LIS1 and Adenomatous Polyposis Coli Timothy Joshua Hines University of South Carolina

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Regulation of Cytoplasmic Dynein by LIS1 and Adenomatous Polyposis Coli Timothy Joshua Hines University of South Carolina University of South Carolina Scholar Commons Theses and Dissertations 2018 Regulation Of Cytoplasmic Dynein By LIS1 And Adenomatous Polyposis Coli Timothy Joshua Hines University of South Carolina Follow this and additional works at: https://scholarcommons.sc.edu/etd Part of the Biological and Chemical Physics Commons Recommended Citation Hines, T. J.(2018). Regulation Of Cytoplasmic Dynein By LIS1 And Adenomatous Polyposis Coli. (Doctoral dissertation). Retrieved from https://scholarcommons.sc.edu/etd/4873 This Open Access Dissertation is brought to you by Scholar Commons. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. REGULATION OF CYTOPLASMIC DYNEIN BY LIS1 AND ADENOMATOUS POLYPOSIS COLI by Timothy Joshua Hines Bachelor of Science Appalachian State University, 2012 Bachelor of Arts Appalachian State University, 2012 Submitted in Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy in Biological Sciences College of Arts and Sciences University of South Carolina 2018 Accepted by: Deanna S. Smith, Major Professor Jeff Twiss, Committee Chair Shannon Davis, Committee Member Sofia Lizarraga, Committee Member David Mott, Committee Member Cheryl L. Addy, Vice Provost and Dean of the Graduate School © Copyright by Timothy Joshua Hines, 2018 All Rights Reserved. ii DEDICATION This work is dedicated to my parents who have supported me in every endeavor I’ve attempted. Well, at least the ones that didn’t get me into trouble. iii ACKNOWLEDGEMENTS First of all, I would like to thank Deanna for giving me the opportunity to carry out research in her laboratory and for her guidance and funding over the years. She’s been a great mentor, from when I first joined her lab and had never heard of dynein, all the way to my defense and hunt for postdocs. Her lighthearted attitude carried me through the trials of graduate school and I appreciate the support she’s given me throughout. I would also like to thank Jeff Twiss who has acted as a co-mentor to me in Deanna’s absence and was frequently available for guidance and advice. Finally, I would like to thank my other committee members Shannon Davis, Sofia Lizarraga, and David Mott for all their work and guidance over the years. I would like to thank all of the people in and around the lab before and after me, including; Feng and Xu Gao (no relation!), Liang “Leo” Shi, Subhshri Sahu Michael Alexander, Zak Roth, and Meghann Lange. Thank you to several members of the Twiss lab, particularly Joon Lee, Amar Kar, Pabi Sahoo, Ashley Kalinski, Liz Thames, and Sharmina Miller-Randolph who have taught me techniques and given me valuable advice for my experiments. Tia Davis deserves a huge thank you for all her hard work over many years behind the scenes in maintaining our mouse colony. I would like to thank my parents, Rick and Loree, my brother Charlie, and my sister Kiersten, and my best friend Richard, for all they have done over the iv years in supporting me. I would like to thank all my other friends and family who I have not explicitly mentioned, as well. v ABSTRACT Cytoplasmic dynein 1 (dynein) is a microtubule motor that plays a role in mitosis, cell migration, and minus-end directed microtubule-based transport. The lissencephaly protein, Lis1, and its binding partner, Ndel1, are critical regulators of cytoplasmic dynein (Niethammer et al., 2000). In humans, haploinsufficiency of Lis1 leads to lissencephaly, a devastating developmental neurological disorder characterized by severe brain malformation, leading to cognitive and motor defects, and progressively worsening seizures (Dobyns et al., 1993). While Lis1 is known to play a role in regulating dynein-dependent functions such as neuronal migration and mitotic spindle orientation during development, the protein is still highly expressed in the adult mouse nervous system, suggesting an important postdevelopmental role in neurons. Indeed, it has been established that Lis1 and Ndel1 regulate dynein-dependent axonal transport in mature neurons (Niethammer et al., 2000; Smith et al., 2000; Pandey and Smith, 2011; Klinman and Holzbaur, 2015). To further elucidate the importance of Lis1 in the adult nervous system, we generated a tamoxifen-inducible Lis1 knockout (KO) mouse to remove the gene post-developmentally. Using an actin promoter to drive expression of a tamoxifen-inducible cre recombinase, homozygous Lis1 KO caused the rapid onset of neurological symptoms, such as hind limb clasping and spinal kyphosis. Administration of tamoxifen resulted in dose-dependent onset of a severe phenotype, which correlated with the extent of recombination observed vi in the midbrain/hindbrain. Chromatolysis, a sign of axonal dysfunction, was observed in neurons of brainstem cardiorespiratory nuclei of Lis1 KO animals. Additionally, transport defects, axonal swellings, and altered neurofilament distribution were observed in cultured DRG neurons from Lis1 KO mice. Restricting Lis1 KO to cardiomyocytes resulted in no observed symptoms, indicating loss of Lis1 in the heart is not the cause of the Lis1 KO phenotype. Thus my work suggests that Lis1 plays a vital role in autonomic neurons and disrupted axonal transport is the primary cause of the Lis1 KO phenotype. Dynein has also been shown to interact with adenomatous polyposis coli (APC). This interaction is regulated by insulin signaling, specifically through phosphorylation by glycogen synthase kinase 3 (GSK3) (Gao et al., 2015; Gao et al., 2017). In wild-type (WT) cells, inhibition of GSK3 causes release of dynein from APC, leading to accumulation of dynein at the centrosome. In cells with the multiple intestinal neoplasia (MIN) mutation of the APC gene, GSK3 is unable to regulate the dynein-APC interaction. Using western blot analysis, I found that the insulin-signaling pathway remains functional in MIN cells. Since APC binds to microtubules, which are the tracks for dynein-dependent transport, I looked for changes in overall microtubule morphology and posttranslational modifications. No difference was observed in microtubule morphology, but there was less detyrosinated tubulin in MIN cells. However, this is unlikely to cause the observed phenotype, as dynein motility is decreased on detyrosinated microtubules (McKenney et al., 2016). Finally, using coimmunoprecipitation, I found that dynein interacts with the C-terminus of APC, which is absent in MIN vii cells, and not the N-terminus. My work indicates that the absence of the C- terminus of APC, and not alterations of the microtubule cytoskeleton or insulin- signaling pathway, is responsible for the inability of GSK3 to regulate dynein in MIN cells. viii TABLE OF CONTENTS DEDICATION ........................................................................................................... iii ACKNOWLEDGEMENTS ............................................................................................ iv ABSTRACT ............................................................................................................. vi LIST OF FIGURES ..................................................................................................... x CHAPTER 1: GENERAL INTRODUCTION ...................................................................... 1 CHAPTER 2: NEURONAL TRANSPORT AND SPATIAL SIGNALING MECHANISMS IN NEURAL REPAIR ................................................................. 13 CHAPTER 3: AN ESSENTIAL POSTDEVELOPMENTAL ROLE FOR LIS1 IN MICE ............... 48 CHAPTER 4: REGULATION OF DYNEIN BY APC AND GSK3 ........................................ 83 CHAPTER 5: DISCUSSION AND CONCLUSIONS .......................................................... 97 REFERENCES ...................................................................................................... 104 APPENDIX A: PERMISSIONS TO REPRINT ............................................................... 123 ix LIST OF FIGURES Figure 2.1 Schematic of neural trafficking in mature, injured, and regenerating axons ........................................................................... 47 Figure 3.1 Lis1 protein is expressed in adult mouse tissues ............................... 76 Figure 3.2 Lis1 knockout impacts axonal function in adult mouse DRG neurons ............................................................................... 77 Figure 3.3 Lis1 knockout via IP tamoxifen injection in adult mice results in a severe phenotype .................................................................. 78 Figure 3.4 Cre-dependent recombination in the brain after tamoxifen injection .. 79 Figure 3.5 Both neurons and glia show evidence of cre-dependent recombination ................................................................... 80 Figure 3.6 Brainstem neurons in Lis1 knockout mice exhibit signs of chromatolysis .............................................................................. 81 Figure 3.7 Comparing the effect of Lis1 knockout in brainstem and heart .......... 82 Figure 4.1 Insulin signaling pathway remains functional in cells with APC MIN mutation ............................................................................ 93 Figure 4.2 Overall microtubule cytoskeleton is unaffected by MIN mutation ....... 94 Figure 4.3 MIN mutation may affect microtubule posttranslational modifications ................................................................. 95 Figure 4.4 Dynein binds C-terminus, and not N-terminus of APC ......................
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